Para-hydroxycinnamic acid (pHCA) is a high-value, aromatic chemical compound that may be used as a monomer for the production of Liquid Crystal Polymers (LCP). LCPs are used in liquid crystal displays, and in high speed connectors and flexible circuits for electronic, telecommunication, and aerospace applications. Because of their resistance to sterilizing radiation and their high oxygen and water vapor barrier properties, LCPs are used in medical devices, and in chemical and food packaging. Due to its importance as a high value, aromatic chemical compound, pHCA has been chemically synthesized (JP 2004231541; JP 2004149438; U.S. Pat. No. 5,705,618; JP 07017898). However, the chemical synthesis methods are expensive due to the high cost of the starting materials and the extensive product purification required. Moreover, the chemical synthesis methods generate large amounts of unwanted byproducts.
Biological production of pHCA may offer a low cost, simplified synthetic route. In plants, pHCA (also known as p-coumarate) is made as an intermediate for the synthesis of various secondary metabolites such as lignin [Plant Biochemistry, Ed. P. M. Dey, Academic Press, (1997)] and isoflavonoids. Phenylalanine ammonia-lyase (PAL) converts L-phenylalanine to trans-cinnamic acid (CA), which is then converted to pHCA. Methods of pHCA isolation and purification from plants are known [R. Benrief, et al., Phytochemistry, 47, 825-832; (1998)], however, these methods are time consuming and cumbersome and do not therefore provide an economical alternative to the current chemical synthesis route. PAL enzymes are also found in fungi (Bandoni et al., Phytochemistry 7:205-207 (1968)), yeast (Ogata et al., Agric. Biol. Chem. 31:200-206 (1967)), and Streptomyces (Emes et al., Can. J. Microbiology 48:613-622 (1970)), but not in Escherichia coli or mammalian cells (Hanson and Havir In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic: New York, 1967; pp 75-167).
Some PAL enzymes, in addition to their ability to convert phenylalanine to cinnamate, can accept tyrosine as a substrate (PAL/TAL enzymes). The tyrosine ammonia lyase (TAL) activity of these enzymes directly converts tyrosine to pHCA. PAL/TAL enzymes have been introduced into microorganisms for production of pHCA (U.S. Pat. No. 6,368,837, US20040059103 A1). These engineered microorganisms expressing TAL activity can be used in fermentation processes for production of pHCA. Yield and rate of pHCA production is enhanced at high pH, between 8 and 11, so that a two-step fermentation and production process is used for efficient pHCA production (US 20050260724). Extended reuse of the cells containing TAL enzyme, the biocatalyst, in pHCA synthesis would further enhance the economical productivity of the process. However, at the high pH used in the pHCA synthesis reaction, the engineered bacterial biocatalyst cells undergo lysis making reuse of the biocatalyst or catalytic enzyme difficult due to recovery issues.
Biocatalysts have been immobilized to provide a more stable or more easily manipulated enzyme source for enzyme catalyzed processes (Lindhardt, R. J., Immobilized biocatalysts. 1987. Appl. Biochem. Biotechnol., 14, 121-145). Biocatalysts with PAL enzyme activity that are used for converting trans-cinnamic acid and ammonia to L-phenylalanine (reverse of the physiological reaction) have been immobilized. The commonly used PAL biocatalysts for phenylalanine production are yeasts such as Rhodotorula glutinis (also called Rhodosporidium toruloides). Rhodotorula glutinis cells with PAL enzyme activity were immobilized and used in production of L-phenylalanine methyl ester (D'Chuna et. al Enzyme and Microbial Technology 19:421-427 (1996)). Immobilization was carried out using various agents including immobilization in calcium alginate beads, agarose beads, and PEI-coated calcium alginate beads. In all cases, L-phenylalanine methyl ester production was decreased following immobilization.
U.S. Pat. No. 4,562,151 discloses a process for synthesis of L-phenylalanines using R. glutinis cells expressing PAL that are immobilized within glutaraldehyde (GA) cross-linked polyethyleneimine (PEI) coated alginate beads. The beads were prepared according to a method of Birnbaum et al. (Biotechnology Letters 3:393-400 (1981)), where PEI is added before GA is added and GA is introduced as a 1% (v/v) solution. In the disclosed synthesis process of U.S. Pat. No. 4,562,151, a polyhydric alcohol or polyethelene glycol-(400) is needed to desensitize the PAL enzyme, enhance the rate of reaction, and inhibit inactivation of the PAL enzyme which otherwise occurs after 12 hours.
L-phenylalanine production by immobilized mutant Rhodotorula rubra yeast cells with high PAL activity is described in Evans et. al. (Biotechnology and Bioengineering, 30, 1067-1072 (1987)). Cells were immobilized in beads of sodium alginate, polyethylene glycol (PEG), glycerol, glutamate, and sorbitol that were hardened with glutaraldehyde. PEG was needed to stabilize the biocatalyst, along with sorbitol and continuous nitrogen purging to remove oxygen. The reaction rate decreased after the first 10 hours and was further reduced in subsequent runs with reused biocatalyst.
Though enzymes with TAL activity as well as PAL activity are used in biocatalysts for production of pHCA, both the biocatalyst and the reaction have major differences with those used in phenylalanine production. Rather than yeast biocatalysts, bacterial cells engineered for high levels of accumulation of TAL activity are desirable as the biocatalyst. Rather than highly soluble substrates, the tyrosine substrate for TAL-mediated synthesis of pHCA is highly insoluble, and partially crystalline at concentrations used in production reactions. These factors provide additional challenges to preparing an immobilized TAL biocatalyst for use in pHCA production.
Efficient and economical production of pHCA would benefit from the use of immobilized cells having TAL activity that maintain high levels of activity and physical mechanical stability for a prolonged period of time under the high tyrosine solids reaction conditions, allowing use in multiple, extended production runs.